Journal of the Royal Society of Western Australia, 85:181-182, 2002 Podocyte complexity and terrestriality in frogs H Y Chan, J E O’Shea, P C Withers & T Stewart Zoology, School of Animal Biology M092, University of Western Australia, Crawley WA 6009 Figure 1. High resolution photomicrographs of Bufo marinus (Manuscript received June 2002; accepted November 2002) podocytes showing glomerulus within a Bowman’s capsule (left; scale bar 50 µm) and podocytes on the glomerular capillaries Abstract. We extend Richter & Splechtna’s (1996) study (right; scale bar 10 µm). of the morphology of amphibian podocytes with respect to habitat aridity and water regulation, and describe the perfused and fixed South African clawed toad (Xenopus method for calculating complexity index of podocyte laevis) was obtained from other experiments. Frog pedicels. Our results show that Notaden nichollsi, the most kidneys were perfusion-fixed with 4% paraformaldehyde terrestrial species examined so far, has the highest in cacodylate buffer and post-fixed with osmium- podocyte complexity. The relationship between podocyte thiocarbohydrazide (Richter & Splechtna 1996). They complexity and habitat suggests that glomerular were then mounted on SEM stubs and critical point filtration rate is an important parameter related to habitat dried. A Philips 505 scanning electron microscope was aridity and water regulation in frogs. used to locate glomeruli on the kidney surface, then a Field Emission Scanning Electron Microscope JSM 6300F was used for higher resolution imaging. Introduction Podocytes on the surface of glomeruli in the kidney of Amphibians are found in habitats ranging from fully frogs were difficult to locate, and it was also difficult to aquatic to fully terrestrial and sometimes extremely arid. obtain photographs of the podocytes with sufficient The permeable skin of most amphibians allows resolution for clear definition of the branching patterns. substantial trans-cutaneous water and solute flux, and Nevertheless, we obtained photomicrographs (e.g. Fig 1) frogs have an effective water regulation system for the calculation of podocyte complexity for three comprising a very distinct lymphatic system, kidneys species, the aquatic Xenopus laevis (n =1 individual, 3 and bladder (Steen 1929; Ewer 1952; Bentley et al. 1958; glomeruli, 5 podocytes), the mesic terrestrial Bufo marinus Middler et al. 1968; Bentley 1969; Shoemaker & Nagy (n = 1; 1 glomerulus, 3 podocytes) and the arid terrestrial 1977; Shoemaker et al. 1992). Notaden nichollsi (n = 1; 1 glomerulus, 2 podocytes). Water regulation by frogs is determined in part by Podocyte complexity index glomerular filtration rate (GFR), the rate of water entry Richter & Splechtna (1996) did not provide any detail into the lumen of the renal tubules (Heller 1950). GFR is of the methodology for calculating the index for determined morphologically by the microstructure of podocyte complexity, but merely cited Laskowski & podocyte cells that cover glomerular capillaries, as well Pohlit (1974), a biophysical book published only in as physiological factors such as plasma hydrostatic and German. So, we describe here our interpretation of the colloid osmotic pressure. The pedicels (foot-like methodology required to replicate Richter & Splechtna’s projections) of podocytes form filtration slits, the number (1996) calculations. and size of which influences GFR. Firstly, the number of branches in each branching Richter & Splechtna (1996) used scanning electron level (from 1 to 4) of a podocyte cell body is determined. microscopy to examine the complexity of podocytes for a Primary level podocyte processes branch out to variety of frogs differing in terrestriality. They secondary level processes, and then to tertiary and distinguished five types of podocyte process systems, quaternary levels. In determining branch levels in each and suggested that podocyte pedicel microstructure was podocyte group, the main branch always retains the correlated with habitat i.e. podocyte complexity increases status of the previous branch level. The main branch is with terrestriality. Aquatic frogs have fewer high- defined as the initial branch extending from the cell branching levels than terrestrial species. We examine body, and with the continuation that provides the podocyte complexity for the Australian frog Notaden maximum branching complexity. nichollsi, and Xenopus laevis and Bufo marinus, using a similar protocol to Richter & Splechtna (1996). After the branching levels are determined, the total number of branch endings is counted, and the fraction (Pi) of branch endings belonging to each branch level is Materials and Methods calculated. For each group, Pi is multiplied by the branch level (Zi), and the complexity index (bit value) for each A desert spadefoot toad (Notaden nichollsi) and a cane podocyte cell body is calculated by summing the PiZi toad (Bufo marinus) were killed by double pithing, and a values. The complexity index is defined such that the higher the bit value, the more complex the structure is. © Royal Society of Western Australia 2002 An example of how to determine the branching levels of 181 Journal of the Royal Society of Western Australia, 85(4), December 2002 Figure 3. Relationship between habitat and podocyte complexity (adapted from Richter & Splechtna 1996). Values from the present study are Xenopus laevis (X; 1.27 bits), Bufo marinus (B; 2.32 bits) and Notaden nichollsi (N; 2.46 bits). Species from Richter & Splechtna (1996) are; 1, Xenopus laevis; 2, Rana ridibunda; 3, Rana esculenta; 4, Bombina variegata scabra; 5, Discoglossus pictus; 6, Rana lessonae; 7, Alytes cysternasii; 8, Bufo regularis; 9, Rana temporaria; 10, Rana dalmatia. between podocyte microstructure, kidney function and water balance for the many and diverse species of anuran amphibians. Acknowledgements: Collection of frogs was under permit from the Department of Conservation and Land Management, and Total number of branch endings = 5 experimentation was under approval from the Animal Ethics Committee of the University of Western Australia. We thank the Neurobiology Research Group of the Department of Zoology (UWA) for perfused and Pi Z i PiZi 1o 1/5 1 1/5 fixed Xenopus laevis specimens. 2o 3/5 2 6/5 3o 1/5 3 3/5 References 10/5 = 2.0 bit Bentley PJ, Lee AK & Main AR 1958 Comparison of dehydration and hydration of two genera of frogs (Heleioporus and Figure 2. Example of calculation of podocyte complexity index Neobatrachus) that live in areas of varying aridity. Journal of for branching level for podocyte pedicels (type III, from Richter Experimental Biology 35: 677-684. & Splechtna 1996) using schematic of primary pedicel branch Bentley PJ 1969 Neurohypophysial function in Amphibia: (1°), secondary branches (2°) and tertiary branch (3°). hormone activity in the plasma. Journal of Endocrinology 43: 359-369. the processes of podocytes is shown in Fig 2. The Ewer RF 1952 The effects of posterior pituitary extracts on water podocyte complexity index for an individual podocyte is balance in Bufo carens and Xenopus laevis, together with some determined by taking the average SP Z value for several general considerations of anuran water economy. The i i Journal of Experimental Biology 29:429-439. randomly selected podocyte cell bodies. Heller H 1950 The comparative physiology of the neurohypophysis. Experientia 6:368-376. Results and Discussion Laskowski W & Pohlit W 1974 Biophysik. Bd 1: Struktur, Energie, Information und Bausteine belebter Systeme. The average complexity index obtained for pedicels of George Thieme Verlag, Stuttgart. podocytes from X. laevis was low, at 1.27 bits; it was 2.32 Middler SA, Kleeman C R & Edwards E 1968 Lymph bits for B. marinus; and was highest (2.46 bits) for N. mobilization following acute blood loss in the toad, Bufo nichollsi. The bit value obtained for X. laevis was very marinus. Comparative Biochemistry and Physiology 24:343- close to that of Richter & Splechtna (1996). Direct 353. comparison between the complexity indices obtained in Richter S & Splechtna H 1996 The structure of anuran podocyte this study with those of Richter & Splechtna (1996) shows — determined by ecology? Acta Zoologica 77:335-348. a strong relationship between podocyte pedicel Shoemaker VH & Nagy KA 1977 Osmoregulation in amphibians complexity and terrestriality (Fig 3), presumably and reptiles. Annual Review of Physiology 39:449-571. mediated by variation in glomerular filtration rate. Shoemaker VH, Hillman SS, Hillyard SD, Jackson DC, McClanahan LL, Withers PC & Wygoda ML 1992 Exchange The podocyte pedicel index for N. nichollsi, the most of water, ions, and respiratory gases in terrestrial arid terrestrial amphibian examined so far, is also the amphibians. In: Environmental Physiology of the highest value measured, conforming well with the Amphibians (eds M E Feder & W W Burggren). University of pattern established by Richter & Splechtna (1996). Chicago Press, Chicago, 125-150. Further studies of additional species will clearly be Steen WB 1929 On the permeability of the frogs’ bladder to productive in more fully documenting the relationship water. Anatomical Record 43:215-220. 182.